This invention relates to the production of catechol and precursors thereof by the conversion of biomass-derived carbon sources. More particularly this invention is directed to the biocatalytic conversion of glucose, and other sugars capable of being used in the biosynthesis of aromatic amino acids, to catechol by way of 3-dehydroshikimate and protocatechuate.
Catechol is an exceptionally important molecule used as a starting material in the synthesis of pharmaceuticals, pesticides, flavors, fragrances, solar protectants, and polymerization inhibitors. Annual worldwide production exceeds 20,000 metric tons.
Currently, most commercial production of catechol is based on the reaction of phenol with peracids or the metal-catalyzed (Fe.sup.+2, Co.sup.+2) reaction of phenol with hydrogen peroxide. Catechol is also produced from the distillation of coal tar. However, these procedures are not entirely desirable given that they use non-renewable, fossil fuel-based starting materials and yield complex mixtures of catechol with aromatic by-products such as hydroquinone. Moreover, these reactions require high temperatures and involve the use of environmentally sensitive materials. Other routes to catechol include alkaline hydrolysis of o-chlorophenol and Fries peroxide rearrangement of salicylaldehyde, but these are no longer commercially viable.
Microbial biosynthesis has also been used for producing catechol, albeit not commercially. Microbial biosynthesis requires the identification of microbes capable of converting starting materials to catechol. Although some such microbes have been identified, the substrates for their biocatalytic activity are benzene or phenol. Benzene, a known carcinogen, poses significant environmental risks. Furthermore, benzene and its derivatives (such as phenol) are produced from fossil fuels, a non-renewable resource. Thus, to date, microbial biosyntheses of catechol have presented no discernible advantage over traditional chemical syntheses from the standpoints of environmental safety and resource consumption.
There are also reports in the literature of the incidental formation of catechol along with other products when certain microbial strains and mutants are cultured in a medium where m-glucose is the carbon source. However, the described strains yield an uncertain and complex mixture produced by way of an undefined biocatalytic pathway.
It would be desirable to provide a synthesis route for catechol which not only avoids reliance on environmentally sensitive starting materials but also makes efficient use of renewable resources. It would further be desirable to provide a synthesis route for catechol which provides catechol as an exclusive product rather than as part of a complex mixture.
The present invention provides methods for the microbial biosynthesis of catechol from readily available carbon sources capable of biocatalytic conversion to D-erythrose 4-phosphate (E4P) and phosphoenolpyruvate (PEP) in microorganisms having a common pathway of aromatic amino acid biosynthesis. One preferred carbon source is D-glucose. Advantageously, D-glucose, and the various other carbon sources useable in connection with the present invention, are non-toxic. Furthermore, they are renewable resources derived from starch, cellulose and sugars found in corn, sugar cane, sugar beets, wood pulp and other biomass resources.
Host microbial organisms suitable for carrying out biosyntheses in accordance with the present invention belong to genera possessing an endogenous common pathway of aromatic amino acid biosynthesis. Preferred host organisms are mutant strains of Escherichia coli genetically engineered to express selected genes endogenous to Klebsiella pneumoniae. One preferred E. coli mutant for use in this invention is E. coli AB2834, an auxotrophic mutant which is unable to catalyze the conversion of 3-dehydroshikimate (DHS), an intermediate along the common pathway, into shikimic acid due to a mutation in the aroE locus which encodes shikimate dehydrogenase.
The common pathway of aromatic amino acid biosynthesis produces the aromatic amino acids phenylalanine, tyrosine, and tryptophan in bacteria and plants. The common pathway ends in the branch point molecule chorismate, which is subsequently converted phenylalanine, tyrosine, and tryptophan by three separate terminal pathways.
Approaches for increasing the efficiency of production of the common pathway have been described in U.S. Patent No. 5,168,056 (issued Dec. 1, 1992), and U.S. patent application Ser. No. 07/994,194 filed Dec. 21, 1992, the disclosures of which are expressly incorporated herein by reference.
In using the genetically engineered mutant host organisms to produce catechol according to this invention, carbon flow directed into aromatic amino acid biosynthesis proceeds along the common pathway to yield elevated intracellular levels of the DHS intermediate, which accumulate due to a mutation which prevents conversion of DHS to chorismate along the common pathway. In a pathway diverging from the common pathway, DHS serves as the substrate for the enzyme 3-dehydroshikimate dehydratase to produce protocatechuate which is thereafter converted to catechol with protocatechuate decarboxylase. Preferably these enzymes are constitutively expressed in the host cell as a result of the transformation of the host cell with recombinant DNA comprising genes encoding those enzymes. Carbon flow thereby is forced away from the common pathway into the divergent pathway to produce catechol.
For example, in the host strain E.coli AB2834, intracellular concentrations of DHS increase due to a mutation in a gene (aroE) which encodes shikimate dehydrogenase. DHS is transformed to catechol along a divergent pathway enabled by transformation of the host cell with expressible genetic fragments encoding DHS dehydratase and protocatechuate decarboxylase and with genes encoding for enzymes which work to commit an increased amount of carbon to the common pathway of aromatic amino acid biosynthesis. The result is a divergent pathway in which carbon flow originally directed into the common pathway of aromatic amino acid biosynthesis is directed to protocatechuate from DHS, and thereafter to produce catechol from protocatechuate. Analysis of the culture supernatants of recombinant mutants of this invention using nuclear magnetic resonance spectroscopy (NMR) demonstrates that catechol accumulates extracellularly as an exclusive product.
Additional objects, features, and advantages of the invention will become apparent to those skilled in the art upon consideration of the following detailed description of preferred embodiments exemplifying the best mode of carrying out the invention as presently perceived.